Drosophila capulet (capt), a homolog of the adenylyl cyclase-associated protein that binds and regulates actin in yeast, associates with Abl in Drosophila cells, suggesting a functional relationship in vivo. A robust and specific genetic interaction is found between between capt and Abl at the midline choice point where the growth cone repellent Slit functions to restrict axon crossing. Genetic interactions between capt and slit support a model where Capt and Abl collaborate as part of the repellent response. Further support for this model is provided by genetic interactions that both capt and Abl display with multiple members of the Roundabout receptor family. These studies identify Capulet as part of an emerging pathway linking guidance signals to regulation of cytoskeletal dynamics and suggest that the Abl pathway mediates signals downstream of multiple Roundabout receptors (Wills, 2002).

Previous studies in Drosophila have shown that although Abl, Ena, and Profilin proteins are expressed broadly during embryogenesis, at later embryonic stages they accumulate at highest levels within the developing nervous system. Capt is abundantly expressed in early stage embryos (e.g., stage 4), consistent with its documented role in oogenesis. In addition to expression in mesoderm and developing gut epithelia, Capt is abundant in the ventral nerve cord (VNC) at stages 12 and 13 when axon pathways are pioneered within the CNS. At late stages (stages 16 and 17), when the last axon pathways are maturing and synaptogenesis is beginning, Capt preferentially accumulates within the VNC. Therefore, Capt, Abl, Ena, and Profilin are coexpressed in neurons at stages important for axonal development (Wills, 2002).

Capt and Abl are able to associate in a physiological setting. Using monoclonal antibodies (mAbs) specific to Abl, it is possible to immunoprecipitate (IP) endogenous Abl from Drosophila S2 cell lysates. When IPs of Abl were analyzed by SDS-PAGE and subsequent Western blot with anti-Capt antisera, endogenous Capt was detected as a protein that coprecipitates with Abl. For further confirmation of Capt protein association, S2 cells were transfected with a cDNA construct encoding full-length capt with a hemoagglutinin (HA)-epitope tag. Anti-HA antibody Western blots of Abl IPs detected a protein of the molecular weight expected for the tagged version of Capt (Wills, 2002).

Genetic experiments suggest that Abl interacts with a number of actin regulatory proteins to control cytoskeletal assembly. Given the functional redundancy observed between CAP and Profilin in yeast, it was thought that Capt and Profilin might participate in some form of protein complex regulated by the Abl kinase. S2 cells were transfected with full-length Drosophila Abl (dAbl), Drosophila Src64 (dSrc), or the truncated mammalian v-Abl and then Capt immunoprecipitations were assayed with anti-Profilin (Chic) and anti-actin antibodies. No significant binding of Capt and Profilin were seen in cells transfected with dSrc or v-Abl or in untransfected controls where endogenous dAbl is expressed at very low levels. However, an association of Capt with Profilin and with actin was observed when dAbl was elevated, suggesting a model where Abl, Capt, and Profilin function together in a cytoskeletal protein complex (Wills, 2002).

The expression and interactions of Capt protein raised the question of whether Capt contributes to the function of the Abl pathway during nervous system development. However, examination of many independent capt allelic combinations that remove zygotic expression without affecting other genes nearby revealed no defects in the embryonic CNS. This is attributed to the large maternal supply of Capt protein visible in the early embryo. Unfortunately, like Profilin null mutations (chickadee), capulet null alleles completely block oogenesis, preventing the use of germline mosaics for the study of zygotic phenotypes in the absence of maternal expression. However, because strong zygotic phenotypes can be induced when mutations in various Abl pathway components are combined with mutations in Abl (e.g., disabled), it was reasoned that zygotic functions of capt might be revealed through genetic interactions (Wills, 2002).

Among the strongest genetic interactions are synthetic phenotypes that arise in transheterozygotes, which lack only one copy of each interacting locus. Heterozygotes that lack one copy of capt or Abl alone show no detectable CNS phenotypes when compared to wild-type strains. However, combination of one capt and one Abl allele results in a distinct axon pathfinding defect (Wills, 2002).

Axons in the Drosophila embryonic CNS are organized into two major groups: longitudinal pathways that extend along the anterior-posterior axis and commissural pathways that carry contralateral projections across the midline. The midline, composed of specialized glial cells, acts as an organizing center that provides secreted growth cone attractants (Netrins) to build commissural pathways and a secreted repellent (Slit) to prevent inappropriate midline crossing. Subsets of longitudinal axons that depend on Slit to maintain their ipsilateral trajectories can be visualized specifically at late embryonic stages (stage 17) with the anti-Fasciclin II (FasII) antibody mAb 1D4; these FasII-positive axons never cross the midline in wild-type embryos (Wills, 2002).

In contrast to wild-type, capt-Abl transheterozygotes display consistent axon guidance defects at the CNS midline. In these double mutants, ipsilateral axon fascicles now ectopically cross, primarily from the most dorsal-medial MP1 pathway. An allelic series of this capt-Abl synthetic phenotype is seen across many different transallelic combinations, showing that the effect is independent of genetic background. No gross defects in the number or fates of postmitotic neurons were detected in any capt-Abl mutants. Although temporal delays were sometimes observed, capt-Abl transheterozygotes did not show any lasting defects in embryonic motor axon pathways (Wills, 2002).

To test whether the midline axon guidance function for capulet is dependent on Capt expression in postmitotic neurons, a wild-type capt transgene was expressed under control of P[elav-GAL4] in a strong capt-Abl background; a 15-fold rescue of the capt-Abl phenotype was observed. Interestingly, a parallel rescue experiment using an N-terminal deletion removing the putative Capt adenylyl cyclase-associated domain provides only a 2.7-fold rescue under the same conditions, despite the fact that the same transgene fully rescues captacuE636 to viability. Thus, capulet and Abl cooperate specifically during midline axon guidance (Wills, 2002).

The failure of the midline gatekeeper function in capt-Abl transheterozygote embryos suggested that Capt might function in the repellent pathway downstream of Slit. To test this genetically, transheterozygotes lacking one allele of capt and one allele of slit were examined. These mutants show a significant increase in the number of midline crossing errors compared to controls. This genetic interaction is seen consistently with multiple alleles of capt. Thus, capt and slit cooperate during midline guidance (Wills, 2002).

To further test the model that capt acts in the repellent pathway, the system of receptors was examined. However, examination of single gene mutations might not be sufficient. This is because the response to Slit is mediated by multiple receptors: Robo, Robo2, and Robo3. Indeed, capt transheterozygotes lacking single alleles in robo, robo2, or robo3 alone show little if any midline phenotype. Yet, when capt alleles are combined with double mutations lacking one copy of robo and robo2 simultaneously, a phenotype almost 2-fold greater than that seen in the robo,robo2 heterozygous embryos is observed. Interestingly, capt/+ does not enhance the phenotype of robo,robo3 heterozygotes, which is already quite strong (Wills, 2002).

As capt activity is further reduced, the interaction with robo2 gets stronger; mutants lacking two copies of capt and one copy each of robo2 and robo3 display penetrant midline phenotypes. Since these allelic combinations are the most severe, they were used for more detailed phenotypic analysis. For example, since the repulsion of growth cones at the midline is dependent on the presence of the midline glia, which secrete the Slit repellent, the midline glia in these mutants were examined with anti-Wrapper antibody, which specifically stains the surface of these glial cells. Midline glia are present in capt-robo2,robo3 mutants, even where axons inappropriately crossed the midline. The first axons in the MP1 fascicle were examined just as they pioneer the ipsilateral pathway early in CNS development. At stage 12, the posterior corner cell (pCC) extends its axon along an anterior trajectory parallel to the midline in order to pioneer the most medial Fasciclin II-positive (MP1) pathway. In capt-robo2,robo3 mutants, pCC axons were sometimes found that had turned toward and crossed the midline at this early stage. This phenotype is similar to that seen in robo alleles (Wills, 2002).

Previous studies of Abl function during midline guidance led to a model where Abl acts to antagonize Robo signaling. However, analysis of capt-Abl transheterozygotes suggests that Abl might play a dual role and also be required for restriction of midline crossing. Consistent with this prediction, examination of several Abl homozygotes reveals an allelic series of midline crossing phenotypes identical to those seen in capt-Abl transheterozygotes. Expression of a wild-type Abl transgene under its endogenous promotor in a strong mutant background rescues the midline crossing phenotype, as does expression of Abl specifically in neurons; however, a kinase-dead transgene was unable to rescue the defect. Like other aspects of Abl function, the midline crossing defects in Abl mutants can be suppressed by dose reduction of its substrate protein Ena or by loss of the receptor protein tyrosine phosphatase Dlar. These observations demonstrate that Abl is required for inhibiting the passage of ipsilateral axons across the midline and suggest that the role of Abl is more complex than previously appreciated (Wills, 2002).

Since analysis of Abl loss-of-function would predict cooperation between Abl and other genes in the repellent pathway, genetic interactions in embryos transheterozygous for Abl and either slit or combinations of mutations in different roundabout genes (ie. slit/+;Abl/+ or robo,robo2/+,+;Abl/+) were assayed. Surprisingly, these embryos displayed striking midline phenotypes far stronger than control genotypes. For example, slit2/+;Abl2/+ transheterozygotes show a 24-fold enhancement of the slit2/+ phenotype. This experiment strongly supports the model that Abl acts positively in the Slit pathway, consistent with the phenotypes of Abl homozygotes and of all the capulet genetic interactions observed (Wills, 2002).

The network of genetic interactions observed suggests that the Abl pathway is involved in signaling downstream of multiple Robo-family receptors. However, previous studies have shown Abl binding to the Robo cytoplasmic domain in vitro is dependent on a peptide motif (CC3) that is not present in Robo2 or Robo3. It was necessary to have an in vivo test for Abl-Robo interactions to explore this issue. Since Abl appears to act in both positive and negative capacities at the embryonic midline, an alternative genetic assay was used to evaluate Abl interaction with the robo gene family. When wild-type Abl is overexpressed in the developing compound eye, under the control of a synthetic glass promotor (GMR-GAL4), a mild rough-eye phenotype was observed. Thus, this Abl phenotype was tested for interactions with various UAS-Robo transgenes (Wills, 2002).

As predicted from loss-of-function analysis, while expression of wild-type Robo alone has little, if any, effect on retinal patterning, the combination of Abl and Robo causes a striking increase in the severity of the Abl gain-of-function eye phenotype. Thus, Robo serves as an enhancer of Abl activity in this kinase-dependent assay. This is also true of Robo2 and of Robo3. These data support the hypothesis that all Robo receptors can engage the Abl signaling pathway. So, is this in vivo interaction dependent on the Robo domains previously shown to recruit Abl and Ena proteins? Interestingly, neither deletion of CC2 nor deletion of CC3 was found to attenuate the Abl-Robo interaction. A UAS-robo transgene lacking the motif CC1 did show a reduction in eye phenotype when combined with UAS-Abl, but the difference was slight (Wills, 2002).

To confirm that Abl can interact with Robo in a CC3 domain-independent fashion during axon guidance, embryos that overexpress Abl and either wild-type Robo(+) or mutant Robo(DeltaCC3) were examined in postmitotic neurons. Abl gain-of-function alone generates two axon guidance phenotypes: (1) ISNb motor axon bypass of ventral target muscles and (2) ectopic midline crossing. Interestingly, coexpression of Abl and either Robo(+) or Robo(DeltaCC3) dramatically enhances the ISNb axon phenotype; however, there was no effect on midline crossing in any of these genotypes. Thus, in vivo, Abl is capable of a functional interaction with all three Robo receptors via some novel mechanism. However, the midline guidance system is specifically refractory to a simultaneous increase in Abl and Robo activities, perhaps due to the dual role of Abl in this context (Wills, 2002).

This study provides compelling evidence that a member of the adenylyl cyclase-associated protein (CAP) family plays a role in the accurate navigation of developing axons. Phenotypic analysis of double mutant embryos demonstrates that Capt cooperates with Abl, Slit, and multiple Roundabout receptors to prevent the inappropriate traffic of axons across the midline choice point. Consistent with published data on the relative contribution of Robo2 and Robo3 to midline repulsion, it has been found that capt and Abl show stronger interactions with robo,robo2 double mutants; however, Abl does appear to interact with all three receptors. The genetic and biochemical interactions observed suggest both that Capt functions directly in the Abl pathway and that this cytoskeletal regulatory pathway is involved in the repellent response to Slit (Wills, 2002).

Detailed studies of the prototypical growth cone repellent CollapsinI/Semaphorin3A have shown that the repellent response involves a collapse of the leading edge structures supported by actin cytoskeleton. Similar results have been seen for members of the Ephrin and Slit protein families. The fact that repellents promote a net disassembly of actin polymer arrays favors the simple model that repellent signaling antagonizes the actin assembly process (Wills, 2002).

Studies of CAP homologs from yeast, Dictyostelium, mouse, pig, and human suggest that the C-terminal actin binding domain acts to sequester monomers to prevent actin polymerization. More recent studies also suggest that human CAP promotes actin disassembly and monomer recycling through interactions with the actin-depolymerizing factor Cofilin. Consistent with an inhibitory role for CAP-family members, studies of epithelial development and oogenesis in Drosophila demonstrate that Capt functions to suppress the hyperassembly of actin microfilaments. Interestingly, a similar function has been ascribed to Abl and Arg during neurogenesis in the mouse. Thus, a model is favored where Abl helps to recruit and regulate CAP activity to inhibit net actin assembly downstream of Robo family receptors (Wills, 2002).

Previous data supported a simple model where Robo recruits Abl and Ena as components in the repellent pathway. In this model, Ena acts as an effector molecule to link Robo to actin assembly and Abl acts purely to antagonize and/or downregulate Robo. While this study confirms that Abl gain-of-function creates ectopic midline crossing, the additional discovery that Capt and Abl cooperate to support the repellent response and that Abl loss-of-function generates ectopic midline crossing suggests that new models are necessary (Wills, 2002).

The fact that Abl is required for midline restriction suggests that Abl plays a dual role in the Robo pathway. There are different models to explain this. As a key enzymatic component in the signaling pathway, Abl may support repellent signaling (by recruiting the necessary actin binding proteins) and also feed back on the receptor (by downregulating through phosphorylation) to adjust the sensitivity of the pathway. This model is attractive because it may explain how growth cones can adapt to different regions within a gradient of Slit. In order for a growth cone to perceive an extracellular gradient (attractive or repellent) over an extended distance, the dynamic range of the response must be continually adjusted. If the receptor system becomes saturated at any point in the gradient, the growth cone will be blind to the extracellular asymmetry at higher concentrations. Conversely, if receptor output is too low, then the signaling differential across the leading edge may be too small to detect the gradient. It has therefore been postulated that gradient guidance will require some form of adaptation to keep the signaling threshold within the appropriate dynamic range as the growth cone moves toward or away from the source. If Abl is part of the repellent response, it would also be an effective source of feedback to help match receptor sensitivity to the gradient conditions. A similar role has been postulated for MAP kinase in the Netrin signaling pathway (Wills, 2002).

The question of exactly how Abl and its signaling partners interface with the Robo receptor family is still unclear. The biochemical data suggest that Abl, Capt, and Profilin may form a large protein complex. However, the genetic interactions between Abl and robo indicate that the CC3 motif is not necessary for a functional link between Abl and Robo. This makes sense because Abl and capulet also interact with robo2, a receptor that lacks both CC2 and CC3 sequences. It is interesting that deletion of motif CC1, which is conserved in all the Drosophila Robo family members, causes a slight attenuation of the robo-Abl interaction in the assay used in this study. CC1 is also the Robo sequence phosphorylated by Abl in vitro (Wills, 2002).

The emerging picture of axon guidance signaling pathways is highly complex. While this may be required to coordinate the many cell biological events that underlie directional specificity during cell motility, it is also possible that this property provides greater opportunity for signal integration. In this light, the potential link between Capulet and adenyyl-cyclase is intriguing. Cyclic nucleotides (cAMP and cGMP) have potent modulatory effects on axon guidance responses in vitro . Although the rescue experiments show that the N-terminal region of Capulet equivalent to the cyclase-interacting domain of other CAP family proteins is not absolutely required for axon guidance function, the reduced rescue activity of this mutant is consistent with cyclase playing a modulatory role in the repellent pathway (Wills, 2002).

DEVELOPMENTAL BIOLOGY

Uniform act up mRNA expression is observed in early embryos, probably reflecting maternally contributed RNA. During gastrulation, the RNA appears to be concentrated at sites where cells invaginate into the embryo. Later, expression is strongest in the central nervous system. Embryos homozygous for the P element insertion l(2)06955 show greatly reduced transcript levels at this stage. In the eye disc, CAP RNA expression appears strongest anterior to the morphogenetic furrow. Because no mutant phenotypes are seen in acu mutant cells posterior to the furrow, it is likely either that acu functions early in development but the effects of its absence are still apparent later, or that the CAP protein perdures in cells that are no longer transcribing the gene (Benlali, 2000).

Actin-Capping Protein and the Hippo pathway regulate F-actin and tissue growth in Drosophila

The conserved Hippo tumor suppressor pathway is a key kinase cascade that controls tissue growth by regulating the nuclear import and activity of the transcription co-activator Yorkie. This study reports that the actin-Capping Protein αβ heterodimer, which regulates actin polymerization, also functions to suppress inappropriate tissue growth by inhibiting Yorkie activity. Loss of Capping Protein activity results in abnormal accumulation of apical F-actin, reduced Hippo pathway activity and the ectopic expression of several Yorkie target genes that promote cell survival and proliferation. Reduction of two other actin-regulatory proteins, Cofilin and the cyclase-associated protein Capulet, cause abnormal F-actin accumulation, but only the loss of Capulet, like that of Capping Protein, induces ectopic Yorkie activity. Interestingly, F-actin also accumulates abnormally when Hippo pathway activity is reduced or abolished, independently of Yorkie activity, whereas overexpression of the Hippo pathway component expanded can partially reverse the abnormal accumulation of F-actin in cells depleted for Capping Protein. Taken together, these findings indicate a novel interplay between Hippo pathway activity and actin filament dynamics that is essential for normal growth control (Fernández, 2011).

The Hippo pathway has emerged as a crucial regulator of tissue size in both Drosophila and mammals. In Drosophila, the Hpo pathway comprises a kinase cascade in which the Hpo kinase binds the WW domain adaptor protein Salvador (Sav) to phosphorylate and activate the Warts (Wts) kinase. Wts, in turn, facilitated by Mats, phosphorylates and prevents nuclear translocation of the transcriptional co-activator Yorkie (Yki). This leads to transcriptional downregulation of target genes that positively regulate cell growth, survival and proliferation, including the Drosophila inhibitor of apoptosis protein 1 (Diap1; thread - FlyBase), expanded (ex), Merlin (Mer) and wingless (wg) in the inner distal ring, within the proximal wing imaginal disc. The upstream components Ex, Hpo and Wts are also thought to regulate Yki through a phosphorylation-independent mechanism, by directly binding to Yki, sequestering it in the cytosol and thereby abrogating its nuclear activity (Fernández, 2011).

Multiple upstream inputs are known to regulate the core Hpo kinase cassette at various levels. Thus, the atypical cadherin Fat was identified as an upstream component of the Hpo pathway and was proposed to transduce signals from the atypical cadherin Dachsous (Ds) and Four-jointed (Fj), a Golgi-resident kinase that phosphorylates Fat and Ds. Moreover, the two Ezrin-Radixin-Moesin (ERM) family members, Ex and Mer have been reported to lie upstream of the Hpo pathway. Mer and Ex can heterodimerize and are believed to exert their growth suppression activity by activating the Hpo kinase. However, how the different inputs that feed into the core kinase cassette are coordinated to regulate Yki activity is unknown (Fernández, 2011).

ERM proteins form a structural linkage between transmembrane components and actin filaments (F-actin). For instance, mammalian Mer binds numerous cytoskeletal factors, including actin, and appears to act as an inhibitor of actin polymerization. Interestingly, the Merlin-actin cytoskeleton association is required for growth suppression and inhibition of epidermal growth factor (EGFR) signaling. Moreover, F-actin depolymerization promotes activation of the Hpo orthologs MST1 and MST2 in mouse fibroblasts (Densham, 2009). These observations suggest a role for F-actin dynamics in modulating Hpo pathway activity (Fernández, 2011).

Actin filament growth, stability and disassembly are controlled by a plethora of actin-binding proteins. Among these, the Capping Protein (CP) heterodimer, composed of α (Cpa) and β (Cpb) subunits, acts as a functional heterodimer to restrict the accessibility of the filament barbed end, inhibiting addition or loss of actin monomers (Cooper, 2008). In Drosophila, mutations in either cpa or cpb, lead to accumulation of F-actin within the cell and give rise to identical developmental phenotypes that are tissue specific. In the wing blade (BL), the most distal domain of the imaginal disc, cpa and cpb prevent cell extrusion and death, but they are not required for this function in the most proximal domain, the prospective body wall and the hinge wing imaginal disc (Janody, 2006). The Cofilin homolog Twinstar (Tsr) and the Cyclase-associated protein Capulet (Capt) also restrict actin polymerization: Tsr severs filaments and enhances dissociation of actin monomers from the pointed end, whereas Capt sequesters actin monomers, preventing their incorporation into filaments (Fernández, 2011).

This study investigated the relationship between the control of the actin cytoskeleton and Hpo pathway activity. Actin-binding proteins CP and Capt, but not Tsr, were shown to enhance Hpo signaling activity. Moreover, a new relationship was uncovered between the Hpo pathway and the machinery that regulates F-actin, and it was revealed that Hpo signaling activity, like CP, limits F-actin accumulation at apical sites independently of Yki. Finally, it is proposed that regulation of an apical F-actin network by Hpo signaling activity and CP sustains Hpo pathway activity, thereby limiting Yki nuclear import and the activation of proliferation and survival genes (Fernández, 2011).

This report shows an interdependency between Hpo signaling activity and F-actin dynamics in which CP and Hpo pathway activities inhibit F-actin accumulation, and the reduction in F-actin in turn sustains Hpo pathway activity, preventing Yki nuclear translocation and upregulation of proliferation and survival genes (Fernández, 2011).

It is suggested that Ex prevents apical F-actin accumulation through Hpo signaling activity but independently of Yki. ERM proteins can form a structural linkage between transmembrane components and the actin cytoskeleton. Mammalian Mer appears to act as an inhibitor of actin polymerization. Moreover, the Mer-actin cytoskeleton association has a crucial role for growth suppression and inhibition of EGFR signaling. In Drosophila, Mer and Ex are structurally related and appear to have partially redundant functions but vary in their requirement depending on the tissue or developmental stage. In imaginal discs, loss of ex shows stronger phenotypes when compared with those of Mer. Ex might also have a stronger requirement on F-actin dynamics, as loss of ex, but not that of Mer, triggered F-actin accumulation. Surprisingly, loss of hpo, sav, mats or wts also triggered apical F-actin accumulation. Ex is likely to affect F-actin through activation of the Hpo kinase cassette because in most ex mutant clones, overexpressing hpo suppressed F-actin accumulation. Some clones seemed to contain increased F-actin. However, these clones also constricted apically, suggesting that the effect on F-actin levels results from a reduction of the apical cell surface and that in the absence of ex, differential activity of overexpressed hpo triggers cell shape changes. Together, these observations argue that Ex prevents apical F-actin accumulation through Hpo signaling activity but independently of Yki (Fernández, 2011).

Loss of Hpo pathway activity or CP triggerw apical F-actin accumulation. Ex localizes to the sub-apical region of epithelial cells, and colocalizes with an HA-tagged form of Cpa, Ex, Hpo, Sav and Wts all interact with each other through WW and PPXY motifs (Oh, 2009; Reddy, 2008). Therefore, a pool of Hpo, Sav and Wts, localized at apical sites, could directly regulate an actin-regulatory protein. Hpo pathway activity might act downstream of CP on F-actin. In agreement with this, ex overexpression significantly suppresses F-actin accumulation in cells with reduced CP levels. The role of Hpo signaling activity might be to inhibit an actin-nucleating factor, which adds new actin monomers to filament barbed ends free of the capping activity of CP. However, it cannot be excluded that ex overexpression enhances the activity of residual Cpa in cells knocked down using RNAi, nor that Hpo pathway activity acts in parallel to CP on F-actin. Interestingly, although endogenous Ex is upregulated in cells lacking CP, mutant cells still accumulated F-actin. wts mutant clones also upregulated Ex, which, when overexpressed, suppresses growth of wts mutant clones. Therefore, the increased levels of endogenous Ex in cells lacking either CP or wts appears to be insufficient to fully suppress the effects of loss of CP or wts on F-actin and growth, respectively (Fernández, 2011).

The data indicate that CP inhibits Yki nuclear accumulation, activation of Yki target genes, and consequently overgrowth of the proximal wing epithelium. Interestingly, Yki was also found to accumulate in nuclei of wild-type cells adjacent to the clone border. Consistent with a non-autonomous effect of CP loss on Hpo pathway activity, ex-lacZ and diap1-lacZ were upregulated in wild-type cells adjacent to CP mutant clones. However, Ex levels were reduced in wild-type neighboring cells. Cells expressing different amounts of ds and fj also upregulate ex-lacZ, but show reduced levels of Ex. Therefore, loss of CP might affect Fj or Ds levels, creating a sharp boundary of their expression. However, in contrast to clones overexpressing ds or mutant for fj, cell lacking CP also upregulated Ex and Mer inside the mutant clones, indicating that CP also acts cell-autonomously to promote Hpo signaling activity. CP might facilitate Yki phosphorylation by the Hpo kinase cassette as cpa-depleted tissues contain decreased phospho-Yki levels. But, the possibility cannot be excluded that CP also favors the direct binding of non-phosphorylated Yki to Ex, Hpo or Wts (Oh, 2009). Further analysis will be required to elucidate the mechanisms by which CP restricts Yki activity cell autonomously and in wild-type neighboring cells (Fernández, 2011).

The results argue for a constitutive role of CP in Hpo pathway activity, since Yki target genes are upregulated in the whole wing and eye imaginal discs. However, loss of CP did not fully recapitulate the phenotype for core components of the hpo pathway. Despite that, on average, cpb mutant clones located in the proximal wing disc domain were 25% larger than wild-type twin spots; 60% of mutant clones failed to grow. Moreover, in the distal wing epithelium, reducing CP levels induces mislocalization of the adherens junction components Armadillo and DE-Cadherin, extrude and death. Furthermore, in Drosophila, CP also prevents retinal degeneration (Delalle, 2005; Johnson, 2008). This indicates that although loss of CP can, under certain conditions, result in tissue overgrowth due to inhibition of Hpo pathway activity, other factors such as the polarity status, also determine the survival and growth of the mutant tissue. Therefore, in addition to promoting Hpo pathway activity, CP has additional developmental functions in epithelia. However, the possibility cannot be excluded that, like most upstream inputs that feed into the Hpo pathway, CP has a tissue-specific requirement in Hpo pathway activity (Fernández, 2011).

CP, Capt and Tsr all restrict F-actin assembly directly. CP and Capt control F-actin formation near the apical surface and inhibit ectopic expression of Yki target genes, whereas Tsr acts around the entire cell cortex and has no effect on Yki target genes. This argues that Hpo signaling activity is not affected by the excess of F-actin per se but provides significant support to the view that stabilization of an apical F-actin network by CP, Capt and Hpo signaling activity feeds back on the Hpo pathway to sustain its activity (Fernández, 2011).

These findings do not lead to an understanding of where F-actin accumulation intersects Hpo signaling activity because both Hpo signaling activity and F-actin dynamics feedback to each other. For instance, hpo or ex overexpression suppressed growth of CP-depleted cells. But, overexpressed ex and possibly hpo also suppress F-actin accumulation of Cpa-depleted cells. The control of F-actin by Hpo signaling activity and CP might constitute a parallel input, which sustains Hpo pathway activity. Alternatively, F-actin could act upstream of the core kinase cascade, which in turn feeds back to F-actin, to maintain its activity. The identification of additional actin cytoskeletal components that either promote Hpo pathway activity or act downstream of Hpo pathway activity on F-actin would help to discriminate between these possibilities (Fernández, 2011).

How F-actin influences Hpo signaling activity remains to be determined. The apical F-actin network, which regulates the formation and movement of endocytic vesicles from the plasma membrane, might promote the recycling or degradation of Hpo pathway components. Increased F-actin at apical sites would, therefore, affect protein turnover. Alternatively, apical F-actin might act as a scaffold to tether Hpo pathway components apically. In support of this, Ex, Hpo, Sav, Wts and Yki could all interact between each other through WW and PPXY motifs at apical sites (Oh, 2009; Reddy, 2008). Moreover, expression of a membrane-targeted form of Mats enhances Hpo signaling (Ho, 2010). Although Ex and Mer are properly localized in CP mutant cells, other members of the pathway might be mislocalized in the presence of excess F-actin. Interestingly, in mouse fibroblasts, the Hpo orthologs MST1 and MST2 colocalize with F-actin structures and are activated upon F-actin depolymerization (Densham, 2009), suggesting that by tethering Hpo pathway components, F-actin dynamics modulates their activity. Finally, the F-actin network might act as a mechanical transducer. Most of the mechanosensitive responses require tethering to force-bearing actin filaments. Tissue surface tension has been proposed to be a stimulus for a feedback mechanism that could regulate tissue growth. The tension exerted by neighboring cells might be sensed at the cell membrane by the actin cytoskeleton and translated to the regulation of cell proliferation through the Hpo signaling pathway (Fernández, 2011).

Effects of Mutation or Deletion

act up mutations fail to complement the deficiency Df(2L)S3, which removes polytene chromosome bands 21D2-22A1. Available P element insertions in this region were tested for failure to complement acu, and two lethal P element alleles of acu, l(2)06955 and l(2)k01217, that had both been mapped to chromosomal position 21F1-2, were identified. Genomic DNA surrounding these insertions was isolated by plasmid rescue and by subcloning homologous fragments of a P1 phage clone mapping to 21F1-2. Comparison of the sequence of these fragments to the Berkeley Drosophila genome project database allowed a transcript that had been sequenced by the genome project to be aligned onto the genomic DNA surrounding the P elements. Both P elements are inserted in the first intron of this transcript, downstream of an untranslated 85 bp exon (Benlali, 2000).

act up mutant bristles at the wing margin
have abnormal shapes and sizes and are sometimes missing. Zygotic loss of acu function causes lethality with a variable onset, from the embryonic to second instar larval stages. The role of any maternal contribution of acu in embryonic development could not be evaluated, as females with acu mutant germline clones fail to lay eggs, indicating a requirement for acu in oogenesis. These acu phenotypes may be due to an effect on actin polymerization; bristles are actin-based structures, and the actin cytoskeleton is required for transport of molecules from the nurse cells to the oocyte during oogenesis (Benlali, 2000).

A mutation in a novel gene, capulet (cap), was identified in a mosaic screen to isolate mutations that perturb actin organization in germline clones. CAPs have been shown to inhibit actin polymerization in vitro, by sequestering monomeric actin. This actin-binding activity has been mapped to the carboxy-terminal region of CAP; however, a 'verprolin homology'-related domain has been identified in all CAPs, just carboxy-terminal of the polyproline-rich domain. In members of the verprolin/WASP family, this motif binds actin monomers in vitro, but catalyses actin polymerization in vivo. Therefore, in CAP homologues, this region of the protein may be used to facilitate actin binding. As CAP proteins have also been found associated with Abl tyrosine kinase and with adenylate cyclase, it is possible that CAP represents an intermediary in these signal transduction cascades, perhaps altering actin dynamics in response to extracellular cues (Baum, 2000).

To characterize the cap mutant phenotype in detail, actin filaments in wild-type ovaries and in mosaic egg chambers carrying cap germline clones were compared. F-actin organization within the wild-type egg chamber does not appear to change dramatically until stage 10B when actin 'dumping' fibres appear in nurse cells. In cap germline clones, however, the distribution of F-actin appears relatively normal during the very early stages of oogenesis, but becomes highly polarized and dynamic as the egg chamber develops. Ectopic F-actin is first seen at stage 5-6 of oogenesis in a dense structure at the posterior pole of the oocyte. By stages 6-8, ectopic actin filaments appear to shift to the anterior of the oocyte, where they are found close to ring canals. At stage 10B, dumping fibres can be seen forming on schedule in the nurse cells of cap clones. Finally, in eggs, extensive filamentous actin structures form close to the cortex, and ectopic F-actin is visible in the few embryos that are produced. Interestingly, the change in the distribution of F-actin, from posterior cortical to anterior vesicle-like structures, mirrors the reorganization of the microtubule cytoskeleton seen in the wild type during stages 6-8. Repolarisation of the microtubule array is thought to be induced by a signal from posterior follicle cells, dependent upon the prior action of Gurken in the germline. Therefore, to determine whether the same signal also affects actin organization, Gurken function was reduced in the cap mutant background. The localization of ectopic F-actin is unaltered in gurken;cap double germline clones, so further work will be required to identify the cues responsible for the dynamic distribution of actin aggregates in the cap mutant (Baum, 2000).

As ectopic actin structures are formed in cap mutant egg chambers, other F-actin-rich structures are lost. In particular, cortical F-actin underlying the nurse cell membranes disappears prematurely at stages 8-9 of oogenesis. Therefore CAP may simultaneously inhibit actin polymerization at some sites and facilitate the formation of F-actin at others. If the pool of actin within the egg chamber is limited, an alternative hypothesis can be imagined, in which actin filaments are lost from nurse cell cortices to compensate for the formation of actin aggregates within the oocyte. In order to test whether the actin cytoskeleton is similarly polarized in other mutants that have excess accumulation of F-actin, twinstar germline clones were examined. twinstar inhibits actin filament formation in vivo and encodes the Drosophila homolog of an actin-severing protein, cofilin. Although ectopic actin filaments form in twinstar germline clones, as in the cap mutant, ectopic actin aggregates form at sites throughout the early twinstar mutant egg chamber. Therefore, CAP has the specific function of inhibiting actin polymerization within the oocyte (Baum, 2000).

As CAP inhibits actin polymerization within the oocyte, but not in nurse cells, an antibody was generated to Drosophila CAP to see if this localized function is reflected in the wild-type distribution of the protein. This antibody is specific, as it recognizes CAP in tissue extracts. CAP is present throughout the follicle cells and in the germline, but at early stages of oogenesis the protein preferentially accumulates in the oocyte. Later in oogenesis, CAP appears to be localized at the oocyte cortex. Thus, CAP is concentrated in the oocyte, where it functions to inhibit actin accumulation (Baum, 2000).

The genetic screen also identified a mutation in the catalytic subunit of protein kinase A (PKA). Therefore, pka and cap mutant phenotypes in the Drosophila germline were compared. Like the cap mutant, pka germline clones lose nurse cell cortical actin, while simultaneously accumulating ectopic actin structures. In addition, the pka mutant phenotype is sensitive to the dosage of CAP, and actin defects are dramatically enhanced in pka;cap double germline clones. These data suggest that PKA and CAP functionally cooperate in the germline to control actin organization (Baum, 2000).

In cap germline clones, F-actin accumulates in a highly polarized fashion within the egg chamber and oocyte. Thus, whether loss of CAP perturbs other aspects of normal polarity, including the asymmetric localization of mRNAs within the oocyte was investigated. The distributions of bicoid and oskar mRNAs, which localize to anterior and posterior poles of the oocyte, respectively, were examined. Although oskar mRNA is concentrated in one region of the oocyte in over 90% of egg chambers, oskar mRNA is mislocalized in 76% of stage 8-10 cap germline clone egg chambers. Moreover, in 28% of cases, oskar transcripts are localized to the anterior or lateral part of the oocyte. In addition, in 64% of stage-10 egg chambers that maintain correct overall polarity, oskar mRNA has a diffuse distribution and is not tightly focused at the posterior pole. The localization of bicoid transcripts was also examined. bicoid mRNA accumulates at an aberrant site in 65% of cap mutant egg chambers, and is localized to the posterior pole in 36% of stage 8-10 egg chambers. Thus, cap germline clones display two related mRNA polarity defects: (1) although oocytes are able to concentrate oskar and bicoid mRNAs locally within the oocyte, they appear unable to coordinate mRNA polarity with the morphological polarity of the egg chamber; (2) in the majority of egg chambers in which oskar mRNA is correctly transported to the posterior pole of the oocyte, oskar message is not tightly localized at the cortex (Baum, 2000).

Mutations in several actin-related genes disrupt mRNA localization, by inducing microtubule-based cytoplasmic streaming. To test whether loss of CAP also disrupts the distribution of mRNAs by inducing premature cytoplasmic streaming, the movement of yolk particles within cap mutant egg chambers was examined. Interestingly, yolk often fails to form in the oocyte in cap germline clones. Instead, the analysis of yolk autofluorescence in live cap mutant egg chambers reveals abnormal yolk particles accumulating at the nurse cell-oocyte boundary. Therefore CAP may be required for a relatively late step in the formation of yolk granules and/or for the directional transport of yolk into the oocyte. Moreover, in a time-lapse analysis of yolk particles in stage 7-9 cap mutant egg chambers, the movements characteristic of cytoplasmic streaming are not observed. This is not unexpected because streaming disrupts mRNA localization completely, whereas cap mutant oocytes accumulate mRNA determinants at discrete sites. Alternatively, the mRNA and yolk localization defects observed in the cap mutant could result from a misoriented microtubule array. To test this hypothesis kinesin-lacZ (kin-lacZ, a fusion between beta-galactosidase and a plus-end-directed microtubule motor was used to assay microtubule polarity in cap germline clones. In the wild type, kin-lacz translocates to the posterior pole of stage 8-9 oocytes. Within cap germline clones, kin-lacz often becomes concentrated at specific but aberrant sites in the oocyte, indicating that microtubules are polarized but misaligned in the absence of CAP. Interestingly, in cases where kin-lacz is found at the anterior cortex, this altered microtubule polarity is accompanied by a change in morphology of the cap mutant oocyte, which appears to invade the nurse cell cluster. This may in turn contribute to the fusion of nurse cells and oocyte observed in the mutant. Finally, in later egg chambers, kin-lacz appears delocalized, as it does in the wild type following the onset of cytoplasmic streaming. In conclusion, early defects in oskar and bicoid localization in the cap mutant are likely to reflect underlying defects in the microtubule cytoskeleton. Interestingly, pka germline clones exhibit mRNA polarity and yolk defects like those of the cap mutant. These data support the notion that CAP and PKA have related germline functions (Baum, 2000).

To investigate whether CAP has an evolutionarily conserved function to control the spatial organization of F-actin and mRNAs, an examination was made in Saccharomyces cerevisiae (budding yeast), where F-actin structures and a mRNA determinant, ASH1, are asymmetrically localized within the bud. In yeast, in contrast to Drosophila oogenesis, microfilament and microtubule cyoskeletons function independently, and polarity is organized primarily by actin filaments, simplifying the analysis (Baum, 2000).

Yeast cells deleted for cap (capDelta) exhibit several morphological defects. Mutant cells vary in size and shape when compared to the wild type. This reflects unpolarized growth, probably arising from actin-related defects in vesicle targeting. capDelta cells also exhibit a disorganized actin cytoskeleton. The majority of cap mutant cells, however, are still able to generate a polar actin organization, with filaments concentrated in the bud. Moreover, the wild-type F-actin distribution appears accentuated in many capDelta cells, implying that, in yeast, CAP prevents hyperpolarization of the actin cytoskeleton, as it does in Drosophila cap germline clones (Baum, 2000).

To visualize cell polarity in yeast, ASH1 mRNA was used as a reporter. In yeast, ASH1 mRNA is a determinant of cell differentiation and is asymmetrically localized by myosin motors tracking along polar actin cables. Upon cell division, the daughter cell derived from the bud inherits ASH1 message, preventing it from switching mating type. Therefore, using green fluorescent protein (GFP) to label ASH1 mRNA, (gASH1), dynamic cell polarity can be visualized. In the wild type, ASH1 mRNA is assembled into a single particle, which is transported into the bud and rapidly localized to the bud tip (gASH1 is found at the tip in ~95% of small buds). Similarly, capDelta cells amass a gASH1 particle, which enters the bud in 85% of cases, demonstrating the presence of actin cables in the mutant. In half of these cells, however, gASH1 is not found at the bud tip. To assess whether this reflects a defect in mRNA movement or in recognition of the bud tip, gASH1 was followed in the buds of living wild-type and capDelta cells using time-lapse confocal microscopy. In the wild type, gASH1 is observed at the bud tip in 75% of frames, moving 18% of the time between snapshots taken at 10 second intervals. In contrast, gASH1 is only present at the bud tip 19% of the time in the cap mutant, changing position between 44% of consecutive frames. Therefore ASH1 mRNA moves excessively in the mutant, and does not become properly anchored. Thus, in yeast, as in the Drosophila oocyte, CAP is not required to establish asymmetries in the distribution of F-actin and mRNAs, but is necessary to define or stabilize wild-type cell polarity (Baum, 2000).

It can be concluded that CAP is a major regulator of actin dynamics in Drosophila, and that CAP is likely to function to inhibit actin polymerization in vivo, as it does in vitro. A striking feature of the cap phenotype is the accumulation of actin filaments at polar sites within the egg chamber. This cannot be explained by differences in the monomeric actin pool in nurse cells versus the oocyte, as G-actin, as measured by DNaseI staining, is equally distributed within the egg chamber, as is profilin. This distribution of actin filaments is peculiar to the cap mutant, because F-actin accumulates at sites throughout the egg chamber in twinstar germline clones. Thus, CAP inhibits actin filament formation at specific cellular sites, possibly in response to signaling events (Baum, 2000).

In yeast and Dictyostelium, cells that lack CAP exhibit clear defects in the control of actin dynamics. In addition, CAP localizes to cortical actin patches in yeast and to the leading edge of migrating Dictyostelium cells, where actin is most dynamic. Although these data clearly implicate CAP in the control of the actin cytoskeleton, they do not reveal the precise nature of the actin defect in the mutant. In yeast, the actin cytoskeleton is perturbed in cells lacking CAP function. The wild-type asymmetric F-actin distribution appears accentuated in many cap mutant yeast cells. In addition, cap mutant cells maintain the capacity to reorganize their actin cytoskeleton in response to an extracellular pheromone cue. Therefore, in both yeast capDelta cells and in Drosophila cap germline clones, the actin cytoskeleton is disrupted in such a way that ectopic actin filaments form in regions of the cell where CAP and F-actin are concentrated in the wild type, in the yeast bud and in the Drosophila oocyte. This leads to the conclusion that CAP has a conserved role in modulation of the distribution of actin filaments. So, by altering CAP activity, cells may be able to alter actin dynamics differently at distinct cellular locations (Baum, 2000).

In both yeast and multicellular eukaryotes, the actin cytoskeleton responds to cell signaling events. Therefore it is interesting to note that homologs of Drosophila CAP have been shown to interact physically with an Abl tyrosine kinase and adenylate cyclase. These latter proteins transduce extracellular cues, in a way that is not fully understood, to remodel the actin cytoskeleton within the growth cones of migrating neurons to facilitate axon guidance. Thus, CAP may constitute part of the machinery that reorganizes the actin cytoskeleton in response to these signals in neurons and in other polarized cells. Interestingly, the genetic screen also identified the catalytic subunit of protein kinase A (PKA), which acts downstream of adenylate cyclase, as a gene required for proper actin organization and oocyte polarity. Since yeast, Hydra and human CAPs have been shown to facilitate the activation of adenylate cyclase, CAP and PKA may be elements of a conserved signal transduction pathway. The phenotypic similarities shared by cap and pka germline clones suggest that CAP and PKA act together in the Drosophila female germline. Given this interaction, CAP could be a substrate for PKA, or could facilitate the activation of adenylate cyclase upstream of PKA. Alternatively, because a reduction in both CAP and PKA activity leads to a more severe phenotype, the two genes may act in parallel pathways. CAP and PKA are, however, unlikely to be essential components in a common signal transduction pathway in Drosophila because no evidence is found for related CAP and PKA functions in somatic tissues (Baum, 2000).

In existing mutants known to perturb the germline actin cytoskeleton, oocyte polarity is either unaffected or completely disrupted. Therefore, whether oocyte polarity is altered in the cap mutant was investigated by examining the localization of both bicoid and oskar mRNAs. When compared to other known mutants, cap germline clones exhibit novel mRNA polarity defects (although similar defects are exhibited by pka null germline clones). First, cap mutant oocytes are able to localize mRNAs to discrete areas within the oocyte, but the sites of mRNA deposition do not respect the existing morphological axes of the egg chamber. Second, in the majority of stage-10 egg chambers with the correct polarity, oskar mRNA is observed in a shallow gradient, as if diffusing away from the cortex at the posterior pole. Thus, CAP seems to be required, both to coordinate mRNA localization with the axial polarity of the egg chamber, and to tether mRNAs to the cortex. Because microtubules are thought to mediate the transport of mRNAs to opposite poles of the oocyte in the wild type, the defect in oocyte axial polarity in the cap mutant may result from defects in the underlying microtubule cytoskeleton. cap germline clones frequently contain a misoriented microtubule array, with plus ends focused at the anterior cortex. This altered microtubule polarity is therefore probably responsible for the mislocalization of oskar and bicoid mRNAs at early stages of oogenesis. At later stages, following disassembly of the polar microtubule array, an actin-based structure at the posterior pole of the Drosophila oocyte, dependent on CAP and tropomyosin, may act as a tether to hold oskar mRNA at the cortex (Baum, 2000).

Budding yeast cells, like the Drosophila oocyte, also use directional transport to localize an mRNA determinant, ASH1, to a polar site at the cell cortex. In yeast, CAP is required for the localization of ASH1 mRNA. Labeled gASH1 mRNA is found in the bud in the majority of cap mutant cells, where it remains highly motile, failing to become anchored at the bud tip. Thus, in both yeast and Drosophila cap mutants, overall polarity is disrupted even though individual mRNA determinants are concentrated within discrete regions of the cell. Since CAP binds monomeric actin directly and is required for the proper distribution of F-actin in yeast and Drosophila, the primary defect in the absence of CAP is likely to be a change in actin organization. It is proposed that CAP is required, in both organisms, to establish an actin-based spatial reference point at the cell cortex. This would serve to correctly align a polar microfilament or microtubule array, defining the cell's axis of polarity and allowing mRNAs and other cargo to be transported unidirectionally to the cell poles. Finally, at the pole, an analogous actin-based structure may be used to tether mRNAs to a fixed plasma membrane site (Baum, 2000).

Capt, Ena and Abl act in concert to modulate the subcellular distribution of actin filaments in Drosophila

The actin cytoskeleton orders cellular space and transduces many of the forces required for morphogenesis. Genetics and cell biology have been combined to identify genes that control the polarized distribution of actin filaments within the Drosophila follicular epithelium. Profilin and cofilin regulate actin-filament formation throughout the cell cortex. In contrast, Capulet (Capt), the Drosophila homologue of Adenylyl Cyclase Associated Proteins, functions specifically to limit actin-filament formation catalysed by Ena at apical cell junctions. The Abl tyrosine kinase also collaborates in this process. It is therefore proposed that Capt, Ena and Abl act in concert to modulate the subcellular distribution of actin filaments in Drosophila (Baum, 2001).

Cells within the follicle-cell epithelium have a simple polarized actin cytoskeleton, with microfilaments concentrated at apical adherens junctions. This asymmetric distribution of F-actin is maintained through the pronounced epithelial movements that occur during the development of an egg. Midway through this process, as the epithelium begins to migrate, there is an increase in the level of F-actin in border cells, a group of motile follicle cells that migrate through the germline towards the oocyte. Then, as the columnar epithelium expands to surround the egg at late stages of oogenesis, pronounced parallel actin bundles are observed at the basal cell surface of follicle cells. To identify genes regulating the spatial organization of F-actin, actin filaments were examined in mutant cell clones within the follicular epithelium (Baum, 2001).

Analysis was carried out by exploring the function of Capt in follicle cells. Although large capt-null follicle-cell clones can be generated without disrupting the shape of the epithelium, capt mutant cells exhibit profound defects in their actin cytoarchitecture. Strikingly, ectopic F-actin accumulates at the apical surface of capt clones, close to the germline. By comparison, actin filaments at lateral and basal cortices usually remain unaffected. On occasion, however, F-actin is also lost from the basal surface of capt follicle-cell clones within mature egg chambers. In apical sections through capt mutant cells, actin filaments are first observed accumulating at sites of cell-cell contact. Because this is where adherens junctions are situated in the wild type, it suggests that junctional material might nucleate new F-actin synthesis in the capt mutant (Baum, 2001).

To test the specificity of the capt mutant phenotype, actin organization was analyzed in twinstar (tsr) mutant cells. Because tsr encodes the actin-filament-severing protein cofilin, F-actin accumulates in tsr mutant cells, as it does in the capt mutant. In tsr mutant follicle cells, ectopic actin filaments form at apical, basal and lateral cortices, although F-actin accumulation often seems more pronounced at the basal cell surface. Frequently, tsr mutant follicle cells also exhibit an altered columnar morphology. These data suggest that cofilin functions throughout the cell cortex to catalyse the disassembly of actin filaments, whereas in contrast, Capt functions in a polarized manner to regulate apical actin accumulation (Baum, 2001).

The polar actin organization seen in capt mutant follicle cells might result from an asymmetric distribution of the Capt protein. However, it was found with the use of immunofluorescence that the protein is evenly distributed within the cytoplasm of wild-type follicle cells. Therefore it seems possible that the loss of Capt might trigger apical actin filament formation indirectly, by deregulating the function of another, asymmetrically localized, protein that modulates actin filament formation (Baum, 2001).

In vitro, Capt has been shown to inhibit actin polymerization by sequestering actin monomers, mirroring its role in vivo, where it limits actin-filament formation. Profilin, encoded by the chicadee (chic) gene, is another well-characterized monomeric actin-binding protein that can serve to promote or to inhibit actin polymerization. To determine which of these biochemical activities dominate in follicle cells, the phenotype of chic-null mutant clones was examined. In chic mutant columnar follicle cells, levels of F-actin seem markedly decreased at all cortices, although actin filaments are lost preferentially from the basal cortex. Thus, profilin promotes actin polymerization in follicle cells as it does in the Drosophila germline and in imaginal discs. To test whether profilin is also required for the formation of apical actin aggregates in capt mutant cells, actin filaments were examined in chic capt double-mutant cell. In clones of the double mutant, ectopic actin filaments are not formed. Thus, profilin is required for actin-filament formation at the cortex of wild-type follicle cells and for the synthesis of apical F-actin aggregates in the capt mutant. Interestingly, chic capt double-mutant clones exhibit an additional morphological phenotype not seen in clones of either single mutant. Double-mutant cells lose their columnar morphology and collapse, forming a thin squamous-like layer of cells. The loss of Capt therefore perturbs cell architecture in the chic mutant, even though a corresponding change in the level of cortical F-actin is not observed. Moreover, the chic capt double-mutant phenotype shows that an ordered actin cytoskeleton is likely to be crucial for the proper morphogenesis of the columnar epithelium (Baum, 2001).

Initial analysis of Capt in the Drosophila germline shows that this protein is required for proper oocyte polarity. To determine whether Capt is also required for the establishment and/or maintenance of follicle-cell apical-basal polarity, the localization of microtubules, Crumbs, alpha-spectrin and ß-spectrin was examined in capt clones. With these markers, it is found that capt mutant follicle cells retain many aspects of their wild-type epithelial polarity despite the profound change in actin organization. Thus, it is possible that apical actin-filament formation in capt mutant clones is localized by the action of the cell's apical-basal targeting machinery. Alternatively, proximity to the germline could define the site of preferential apical actin-filament formation in a capt mutant cell. To distinguish between these two possibilities clones were generated within the columnar epithelium that lacked both capt and lethal(2)giant larvae [l(2)gl] gene functions (because l(2)gl is required cell-autonomously for the maintenance of epithelial polarity). In capt l(2)gl double-mutant cells, F-actin is frequently seen accumulating at a single, randomly positioned site within the cell. Thus, although components of the apical-basal targeting machinery are not required to limit actin-filament formation to a single site, they are necessary to target actin-filament formation to the apical surface of capt mutant cells. These data led to a postulate of the existence of a protein that is targeted to apical junctions and that promotes local actin-filament formation after the loss of Capt. Ena was considered a potential candidate (Baum, 2001).

Ena family members are thought to be key regulators of actin-filament dynamics. In mammalian cells they promote actin-filament formation and are localized at adherens junctions and focal contacts. To determine whether Drosophila Ena shares similar properties, the distribution and function of Ena within follicle cells was analysed. In early egg chambers, Ena protein is concentrated at the apical cell cortex of wild-type follicle cells. Subsequently, as the epithelium begins to migrate, the level of Ena increases, particularly within posterior follicle cells and in motile border cells. At this stage the protein remains localized together with F-actin at the apical surface of the epithelium. However, Ena is also observed in punctate cytoplasmic structures that lack coincident actin filaments. These structures are present in follicle cells throughout oogenesis but become more striking as the egg chamber develops. Finally, at late stages of oogenesis, Ena protein becomes concentrated at the basal surface of the follicular epithelium, flanking stress-fibre-like actin filaments. To locate Ena more precisely, the distribution of Ena was compared with that of Armadillo (Arm). Arm is the Drosophila ß-catenin homologue and is localized at apical adherens junctions interconnecting cells within the epithelium. It was found that Ena and Arm localize together apically in wild-type follicle cells. Thus, through much of oogenesis, Ena is concentrated together with F-actin and Arm at follicle-cell adherens junctions, the site of ectopic actin-filament formation in capt mutant cells (Baum, 2001).

As a test of Ena function, follicular ena clones were generated. Cells homozygous for hypomorphic ena alleles (ena210, ena23) lose cortical actin filaments from apical, basal and lateral sites. However, whereas chic clones preferentially lose basal actin filaments, ena mutant cells also exhibit a marked decrease in the amounts of apical F-actin. This might reflect the fact that Ena is concentrated at apical junctions in the wild type, whereas profilin has a broader cellular distribution. It is concluded that Ena facilitates actin-filament formation in Drosophila, much as it does in mammalian cells (Baum, 2001).

In mammalian cells, the overexpression of Ena homologues is able to induce ectopic actin-filament formation. To determine whether Ena can promote excessive actin-filament formation in Drosophila, Ena was overexpressed in follicle cells and, in a separate experiment, in the germline. In both cell types Ena is able to induce the formation of novel F-actin structures at sites where Ena aggregates accumulate. Thus, Ena is likely to be a critical determinant of the subcellular distribution of actin filaments within a cell (Baum, 2001).

Given Ena's important role in the control of epithelial actin organization, ena capt double-mutant clones were generated to test whether Ena is also required for the formation of apical actin aggregates in the capt mutant. Because ena and capt genes are located on opposite arms of chromosome II, this is an experimental challenge. However, the mutants were recombined onto a double FRT chromosome to generate double-mutant cells that were identified by the absence of green fluorescent protein (GFP) and the loss of Capt. F-actin aggregates do not form in the double mutant lacking both Ena and Capt functions, and ena capt clones frequently lose actin filaments. This result places Ena genetically downstream of Capt. Thus, like profilin, Ena is required for the synthesis or nucleation of actin filaments at adherens junctions in the capt mutant.

Given that the Abl tyrosine kinase binds mammalian Capt and antagonizes the function of Ena in Drosophila, whether Abl, like Capt and Ena, might have a role in the control of F-actin organization in the follicular epithelium was tested. Clonal analysis reveals that loss of Abl causes subtle defects in F-actin organization. In abl4 mutant cells, apical actin filaments are often mislocalized, appearing at elevated levels at lateral cell cortices. In addition, abl clones exhibit severe defects in epithelial architecture, with mutant tissue forming a multilayered epithelium close to the posterior pole of the egg chamber and, to a smaller extent, at the anterior pole. A similar phenotype has been described in dlg/l(2)gl/scrib mosaic egg chambers. Because these genes are required for proper epithelial cell polarity, Abl might also regulate the polarity of follicle cells. As a further perturbation of Abl function the heat-shock Gal4 driver was used to express the protein at high levels within the follicular epithelium. Like the abl loss of function, high-level overexpression of Abl also perturbs epithelial architecture, leading to the formation of multiple layers of cells at the posterior pole of the egg chamber. Thus Abl functions to modulate follicle-cell F-actin organization and cell polarity and must be tightly regulated in follicle cells to maintain proper epithelial character (Baum, 2001).

To test genetically for an interaction between Abl and Capt, Abl was expressed at more moderate levels (using the T155 Gal4 driver) in egg chambers containing capt mutant clones. Although a modest overexpression of Abl has little visible effect on wild-type follicle cells, an increase in Abl expression in capt mutant follicle cells has profound effects. Increased levels of Abl protein alter both the level and distribution of actin filaments in capt mutant cells. The formation of large localized F-actin aggregates seems to be suppressed and actin filaments often become more widely distributed around the cell cortex, as observed in abl mutant clones. In addition, in a minority of egg chambers, the combination of Abl overexpression and loss of Capt causes a profound disruption of epithelial morphology. This genetic interaction between capt and Abl implies that the two genes have related functions in the control of epithelial F-actin organization (Baum, 2001).

Finally, having found genetic evidence to suggest that Ena and Abl cooperate with Capt in the control of epithelial F-actin, the distribution was examined of Ena and Abl proteins in capt mutant follicle cells. In the capt mutant, Ena's distribution is altered so that the majority of the protein becomes localized with apical actin filaments. Thus Ena is found tightly associated with apical F-actin both in the wild type and in capt mutant cells. In contrast, significant amounts of Ena are not observed at the apical surface of capt chic double-mutant cells, in which apical F-actin aggregates are not formed. Therefore, both in the wild type and in various mutants, the amount of Ena present at adherens junctions closely parallels the level of apical F-actin. The localization of Abl was also examined in the wild type and in capt mutant tissue. Abl, like Capt, seems to have a diffuse staining pattern within wild-type follicle cells. However, in capt clones, Abl protein becomes concentrated at the apical cell surface, partly localizing with Ena. Thus, a loss of Capt leads to a change in localization of both Ena and Abl. Because these proteins act together with Capt to control the spatial organization of the follicular actin cytoskeleton, their altered distribution is likely to contribute to the generation of the marked capt mutant phenotype (Baum, 2001).

This study has used Drosophila genetics and the follicular epithelium to characterize how various actin-binding proteins act to regulate the spatial organization of F-actin. The results show that actin dynamics are regulated by distinct mechanisms within different domains of a polarized epithelial cell. Capt, Ena and Abl seem to modulate apical actin-filament formation, whereas cofilin and profilin seem to have a more global function, regulating cortical actin-filament dynamics throughout the cell. Moreover, the accumulation of F-actin at apical, basal and lateral sites in tsr mutant follicle cells and the loss of cortical actin filaments in chic mutant cells indicates that cortical actin filaments are turned over continuously throughout the cell. This being so, it is striking that F-actin becomes so highly polarized in the capt mutant (Baum, 2001).

In vitro, Capt has been shown to inhibit actin polymerization, which is consistent with its role in limiting epithelial actin-filament formation. In such assays, Capt seems to block actin polymerization by sequestering actin monomers. However, Capt protein and monomeric actin seem evenly distributed within the follicular epithelium. Because the loss of a uniformly distributed, actin-monomer sequestering protein would not be expected to result in the polarized accumulation of F-actin, it is considered possible that Capt might function by inhibiting the activity of a protein at apical adherens junctions that promote local actin-filament formation. Given Ena's ability to promote F-actin-filament assembly in mammalian cells, it is considered that Ena is a potential target of Capt activity. Cell-biological and genetic analysis of Ena within the Drosophila follicular epithelium supports this idea. Ena is concentrated together with F-actin at apical adherens junctions, and the level of Ena protein closely parallels the extent of local actin-filament formation. These results indicate that Ena has an important role in dictating the spatial organization of the actin cytoskeleton in Drosophila follicle cells. Moreover, Ena is required for the synthesis of apical actin aggregates in the capt mutant, which adds strength to the hypothesis that Capt might inhibit apical actin-filament formation catalysed by Ena. However, it is important to note that Ena is also present in cytoplasmic aggregates that lack concomitant F-actin and that in the wild type, Ena localizes with F-actin primarily at the apical cell cortex. The presence of Ena is therefore not sufficient to induce local actin polymerization, and its ability to catalyse actin-filament formation might be augmented at adherens junctions. Finally, homologues of Ena have been shown to localize to focal contacts and adherens junctions in mammalian epithelial cells in culture, suggesting that Ena might have an evolutionarily conserved function to control actin-filament formation at these sites (Baum, 2001).

Although profilin binds Ena and is required for F-actin formation within the wild type and in capt mutant follicle-cell clones, profilin seems to differ from Ena in two respects: (1) it is found that the overexpression of profilin has little effect on the level or distribution of F-actin; (2) profilin protein is not localized within follicle cells. Therefore profilin might have a general function within cells, facilitating actin-filament formation throughout, whereas Ena catalyses actin-filament formation at specific subcellular sites (Baum, 2001).

Like Capt and Ena, Abl is also required for proper F-actin organization within the follicle-cell epithelium. Interestingly, Abl also exhibits a cell-polarity phenotype reminiscent of that seen in l(2)gl/dlg/scrib mutants. Furthermore, the activity of Abl has a potent effect on the organization of F-actin in follicle cells lacking Capt. This is different from that observed in capt l(2)gl mutant cells because excess Abl generates more diffusely localized ectopic F-actin. The synergistic interaction between Capt and Abl suggests that these two proteins act in the same pathway. However, because Abl alters the organization of actin within capt mutant cells, Abl and Capt are unlikely to be components in a simple linear signalling cascade. One possibility is that Abl modulates the integrity of adherens junctions, where Ena and Capt seem to act. In support of the idea that Capt and Abl have related functions, mammalian homologues of Capt and Abl have been shown to interact physically, indicating that this relationship might be conserved in mammals. Moreover, actin aggregates reminiscent of those seen in the fly capt mutant cells are formed in the neurons of abl mutant mice. Thus Abl might modulate actin-filament formation in multiple organisms and cell types, perhaps by cooperating with Ena and Capt (Baum, 2001).

Bringing these data together, a model is envisaged in an effort to explain the aetiology of the pronounced capt mutant phenotype. Normally, in the columnar epithelium, Ena protein is concentrated at adherens junctions, where it promotes local F-actin synthesis. This activity of Ena is counterbalanced by Capt, which limits the amount of apical F-actin. Excess F-actin therefore forms at apical junctions in capt mutant cells. This newly formed apical F-actin is able to recruit additional molecules of Ena from the cytoplasm, because Ena binds microfilaments, which leads to further actin-filament formation. This explains why, in the absence of apical F-actin aggregates, Ena does not become concentrated at the apical cortex of cells in the capt chic mutant. Thus, the loss of Capt initiates an explosive cycle of local actin polymerization and Ena recruitment at adherens junctions, culminating in the striking polar actin aggregates observed in capt mutant cells. It is speculated that within wild-type epithelial cells, controlled autocatalytic cycles of actin-filament formation of this type might help to limit the accumulation of actin filaments to a single site within a cell. For instance, during the formation of a Drosophila wing hair, a similar process might be required to generate a single bundle of actin filaments at the apical cortex of the epithelium (Baum, 2001).

In Drosophila, Ena and Abl are thought to be part of a signalling pathway that changes local actin polymerization within the growth cones of neurons to guide axonal pathfinding. However, it has not been possible so far to analyse the cytoskeletal consequences of perturbations in Ena and Abl function directly within Drosophila neurons. The easily visible, asymmetric actin cytoskeleton in follicle cells has allowed the definition of cell-biological roles for these actin-regulatory genes. In these cells, Capt, Ena and Abl modulate actin-filament assembly at specific subcellular sites, probably by altering local actin dynamics. Thus, by analogy, these proteins might alter the site of actin-filament formation in response to signalling in neurons. If so, it will be interesting to determine whether similar signals impinge on this putative Capt/Ena/Abl pathway in neurons and in epithelia. Finally, given the fact that Capt, Ena and Abl control actin cytoskeletal organization in multiple tissues and in different organisms, these genes might have a conserved function, acting together to control the distribution of actin filaments in many other types of polarized animal cell (Baum, 2001).

Influence of Notch on dorsoventral compartmentalization and actin organization in the Drosophila wing: genetic interactions with capulet

Compartment boundaries play key roles in tissue organization by separating cell populations. Activation of the Notch receptor is required for dorsoventral (DV) compartmentalization of the Drosophila wing, but the nature of its requirement has been controversial. Additional evidence is provided in this study that a stripe of Notch activation is sufficient to establish a sharp separation between cell populations, irrespective of their dorsal or ventral identities. Cells at the DV compartment boundary are characterized by a distinct shape, a smooth interface, and an accumulation of F-actin at the adherens junction. Genetic manipulation establishes that a stripe of Notch activation is both necessary and sufficient for this DV boundary cell phenotype, and supports the existence of a non-transcriptional
branch of the Notch pathway that influences F-actin. Finally, a distinct requirement has been identified for a regulator of actin polymerization, capulet, in DV
compartmentalization. These observations imply that Notch effects compartmentalization through a novel mechanism, which is referred to as a fence, that does not depend on the establishment of compartment-specific cell affinities, but does depend on the organization of the actin cytoskeleton (Major, 2005).

It has generally been assumed that compartmentalization is effected by the
establishment of differential cell affinities, which result in cells sorting
to their respective sides of a compartment boundary. Although this
paradigm fits well with studies of AP compartmentalization, it is not easy to
reconcile with studies of DV compartmentalization, given that Notch is
activated and required on both sides of the compartment boundary, that neither
mutation nor ectopic activation of Notch causes directed changes in cell
location, that the requirement for Notch is non-autonomous, and that the
requirement for Notch does not depend on the dorsal or ventral identity of a
cell. Models that have proposed that Notch influences DV compartmentalization
by affecting a compartment-specific cell affinity have required that it act in
conjunction with Apterous. The crucial failing of such models is that
they cannot explain how a compartmental separation of cells is achieved by the
ectopic Notch activation associated with a mutation of fng, or
ectopic expression of Fng, Serrate or Delta, as in all of these cases cells on
both sides of the boundary are identical with respect to the presence or
absence of Ap (Major, 2005).

An alternative hypothesis is that Notch activation influences a property or
behavior of cells at the boundary, referred to as a fence, in a way that prevents them from
intermingling. The determination that Notch
signaling effects a polarized elevation of F-actin and Ena supports this
hypothesis, since it demonstrates that Notch can polarize the actin cytoskeleton
in conjunction with its ability to separate cells, and that this influence of
Notch is independent of the dorsal or ventral identity of the cell.
Additionally, the bidirectional and non-autonomous disruptions of the
compartment boundary effected by capt mutant clones are consistent
with the inference that compartmentalization involves an F-actin-dependent
fence. When the fence is broken, cells can intermix in either direction,
irrespective of their DV identity. By contrast, it is not clear how the
non-autonomous affects of capt mutant clones could be reconciled with
models that postulate a compartment-specific cell affinity (Major, 2005).

The possibility of a non-transcriptional influence of Notch on DV
compartmentalization, as suggested above for its influence on F-actin, is
appealing because it could explain the observation that Fng can influence
compartmentalization even when co-expressed with N-intra.
Loss-of-function studies have provided mixed results as to the requirements
for Notch transcriptional pathways in DV compartmentalization. Clones of cells
mutant for a hypomorphic allele of Su(H), Su(H)SF8,
respect the compartment boundary, even though transcriptional targets are
affected. However, this is not a null situation for Su(H),
and it would be predicted that at a minimum, a Notch transcriptional pathway would
be required at the DV boundary to maintain the expression of Notch ligands. Requirements for
transcriptional mediator proteins confirm that transcription is required for
DV compartmentalization, but a role for a transcriptional Notch pathway does not
preclude a parallel role for a non-transcriptional pathway (Major, 2005).

The molecular nature of the compartmentalization fence is not yet clear,
but some possibilities can be suggested. One model is based on the similarity
of the F-actin stripe at the DV boundary to a prominent F-actin cable detected
along the interface between leading edge cells and amnion-serosa cells during
dorsal closure of the Drosophila embryo. The
F-actin cable and associated proteins are thought to help keep dorsal
epidermal cells in register as they move, through actin-myosin-based
contraction and/or influences on the protrusive behavior of filopodia. Similar
processes could maintain a smooth separation between cells at the DV
compartment boundary. Intriguingly, genetic studies have suggested a potential
role for Notch in dorsal closure that does not involve Su(H). The
distinct requirement for a regulator of actin polymerization, capt,
at the DV boundary is consistent with the hypothesis that the elevated F-actin
detected at the DV boundary plays a crucial role in compartmentalization. In
this view, the F-actin cable would be a physical manifestation of the
Notch-dependent separation fence (Major, 2005).

An alternative possibility is suggested by the observations Notch and its
ligands can, at least in cultured cell assays, act as cell adhesion molecules, and that association of Notch with its ligands can promote cleavage of both molecules.
Thus, while loss- and gain-of-function studies of Notch
ligands do not support the possibility that they act as compartment-specific
cell adhesion molecules, it is suggested that cleavage of Notch and/or its ligands might act as a boundary-specific de-adhesion mechanism. Boundary-specific
de-adhesion, rather than compartment-specific adhesion, has been suggested as
a possible mechanism for Eph-Ephrin-mediated cell separation. In
this model, the influence on F-actin might be a secondary consequence of the
primary separation mechanism. Alternatively, because the cytoplasmic domains
of Notch and its ligands have been reported to associate with proteins that
can impinge on actin organization, Notch or
ligand cleavage might be a direct mechanism for modulating F-actin (Major, 2005).

CAP/Capulet (Capt), Slingshot (Ssh) and Cofilin/Twinstar (Tsr) are actin-binding proteins that restrict actin polymerization. Previously, it was shown that low resolution analyses of loss-of-function mutations in capt, ssh and tsr all show ectopic F-actin accumulation in various Drosophila tissues. In contrast, RNAi depletion of capt, tsr and ssh in Drosophila S2 cells all affect actin-based lamella formation differently. Whether loss of these three related genes might cause the same effect in the same tissue remains unclear. Loss-of-function mutant clones were generated using the MARCM or EGUF system whereas overexpression clones were generated using the Flip-out system. Immunostaining were then performed in eye imaginal discs with clones. FRAP was performed in cultured eye discs. This study compared their loss-of-function phenotype at single-cell resolution, using a sheet of epithelial cells in the Drosophila eye imaginal disc as a model system. Surprisingly, it was found that capt and ssh, but not tsr, mutant cells within and posterior to the morphogenetic furrow (MF) shared similar phenotypes. The capt/ssh mutant cells possessed: (1) hexagonal cell packing with discontinuous adherens junctions; and (2) largely complementary accumulation of excessive phosphorylated myosin light chain (p-MLC) and F-actin rings at the apical cortex. It was further shown that the capt/ssh mutant phenotypes depended on the inactivation of protein kinase A (PKA) and activation of Rho. Although Capt, Ssh and Tsr were reported to negatively regulate actin polymerization, it was found that Capt and Ssh, but not Tsr, share overlapping functions during eye morphogenesis (Lin, 2012; full text of article).

New Dystrophin/Dystroglycan interactors control neuron behavior in Drosophila eye.

The Dystrophin Glycoprotein Complex (DGC) is a large multi-component complex that is well known for its function in muscle tissue. When the main components of the DGC, Dystrophin (Dys) and Dystroglycan (Dg) are affected cognitive impairment and mental retardation in addition to muscle degeneration can occur. Genetic screens have been performed using a Drosophila model for muscular dystrophy in order to find novel DGC interactors aiming to elucidate the signaling role(s) in which the complex is involved. Since the function of the DGC in the brain and nervous system has not been fully defined, this study has analyzed the DGC modifiers' function in the developing Drosophila brain and eye. Given that disruption of Dys and Dg leads to improper photoreceptor axon projections into the lamina and eye neuron elongation defects during development, the function of previously screened components and their genetic interaction with the DGC in this tissue were determined. This study first found that mutations in chif, CG34400, Nrk, Lis1, capt and Cam cause improper axon path-finding and loss of SP2353, Grh, Nrk, capt, CG34400, vimar, Lis1 and Cam cause shortened rhabdomere lengths. It was determined that Nrk, mbl, capt and Cam genetically interact with Dys and/or Dg in these processes. It is notable that most of the neuronal DGC interacting components encountered are involved in regulation of actin dynamics.
These data indicate possible DGC involvement in the process of cytoskeletal remodeling in neurons. The identification of new components that interact with the DGC not only helps to dissect the mechanism of axon guidance and eye neuron differentiation but also provides a great opportunity for understanding the signaling mechanisms by which the cell surface receptor Dg communicates via Dys with the actin cytoskeleton (Marrone, 2011).

The roles that Dys and Dg play in disease have been apparent for some time since their disruption or misregulation has been closely linked to various MDs. Dg depletion results in congenital muscular dystrophy-like brain malformations associated with layering defects and aberrant neuron migration. These defects arise due to extracellular matrix protein affinity problems that influence neuronal communication and result in learning and memory defects. Similar to brain layer formation, the migration of R1-R6 growth cones into the lamina occurs in a similar manner where glia cells that migrate from progenitor regions into the lamina provide a termination cue to innervating axons. In Drosophila Dys and Dg are expressed in the CNS, PNS and visual system and both proteins are required for proper photoreceptor axon guidance and rhabdomere elongation. This work has identified novel components implicated in the process of eye-neuron development. Moreover, it was found that Nrk, Mbl, Cam and Capt genetically interact with Dys and/or Dg in visual system establishment (Marrone, 2011).

The proteins Mbl, Capt, Cam, Robo, Lis1 and Nrk have been shown previously to be associated with the nervous system, and this study has additionally found that mutations in chif, SP2353, CG34400 and vimar cause abnormal photoreceptor axon pathfinding and/or differentiation phenotypes. Lis1 has been shown to bind microtubules in the growth cone, and the human Lis1 homologue is important for neuronal migration and when mutated causes Lissencephaly, a severe neuronal migration defect characterized by a smooth cerebral surface, mental retardation and seizures. This study has found that Lis1RNAi/GMR-Gal4 mutants have abnormally formed lamina plexuses, shortened rhabdomeres, and retinal vacuoles. Chif has been shown to regulate gene expression during egg shell development and is related to a DNA replication protein in yeast. The human ortholog for SP2353 (AGRN) is involved in congenital MD development. Drosophila SP2353 is a novel agrin-like protein that contains Laminin G domains, which makes it a potential new extracellular binding partner for Dg. CG34400 encodes for a protein homologues to human DFNB31 (Deafness, autosomal recessive 31) that causes congenital hearing impairment in DFNB31 deficient people and mouse whirlin, that causes deafness in the whirler mouse. Hearing loss has been as well demonstrated in association with various forms of muscular dystrophy. Vimar has been shown to regulate mitochondrial function via an increase in citrate synthase activity (Marrone, 2011).

Mbl is a Drosophila homologue of the human gene MBNL1. Mutations of this gene cause myotonic dystrophy and are associated with the RNA toxicity of CUG expansion diseases protein. This study shows that Mbl deficiency results in similar phenotypes to Dys and Dg loss of function, and to specifically interact with Dys in axon projections which is in accord with the Dys specific interaction seen in muscle. Dys has multiple isoforms, and the variability of DMD patients to have mental impairment has been linked in part to small Dys isoform mutations, which leads to speculation that Dys is a target for Mbl mediated splicing (Marrone, 2011).

Interestingly, Mbl isoforms have been demonstrated to regulate splicing of α-actinin, which belongs to the spectrin gene superfamily that also includes dystrophins. α-actinin and Capt, the Drosophila homologue of Cyclase-associated protein (CAP) are actin-binding proteins in the growth cone. Capt was first identified in yeast and is highly conserved throughout eukaryotic evolution. The main known function of Capt is to act in the process of actin recycling by working in conjunction with Actin Depolymerization Factor (ADF a.k.a. Cofilin) to help displace Cofilin from G-actin during depolymerization. It has already been reported that ADF/Cofilin has a role in retinal elongation. The actin cytoskeleton is a major internal structure that defines the morphology of neurons, and Capt has already been shown to be required to maintain PNS neuronal dendrite homeostasis in Drosophila via kinesin-mediated transport. Additionally, Capt has been found to lead to excessive actin filament polymerization in the eye disc and to cause premature differentiation of photoreceptors. The rate of axon projection is much slower than the rate of microtubule polymerization during axonal growth, implying that depolymerization/polymerization of actin is important during pathfinding. This study has also shown that Capt interacts with Dys and is necessary for proper projection of photoreceptor axons in the developing brain, and when absent, eyes develop with abnormal rhabdomeres. Furthermore, captRNAi mutants exhibit overgrowth of photoreceptor axons, and it is believed that a possible explanation for this is improper turnover of actin (Marrone, 2011).

Importantly, proteins that can be regulated by Ca2+ to organize actin filament bundles and to promote filament turnover include α-actinin and (ADF)/Cofilin, respectively. Cam functions as an intracellular Ca2+ sensor, and when Ca2+-Cam was selectively disrupted in a subset of neurons in Drosophila embryos, stalls in axon extension and errors in growth cone guidance resulted. Actin turnover is highly regulated by Ca2+ levels, and many proteins are Ca2+-mediated to regulate motility and axon guidance. The results and those from prior studies suggest that Cam is a major functional player of Ca2+ regulation in growth cones. Since it was shown here that mutations in Cam and capt have similar phenotypes in photoreceptor axon pathfinding and rhabdomere development, it is postulated that actin dynamics is the link between these two proteins and the phenotypes described here. Due to the importance of Cam for actin dynamics, its interaction with both Dg and Dys suggests that the DGC coordinates the actin cytoskeleton in the developing eye (Marrone, 2011).

The last gene identified in this work is Nrk. Recently various kinases, channels and other enzymes have been shown to associate with the DGC, although only a few of these interactions have been confirmed in vivo. Since Nrk is a component found to interact with Dys in photoreceptor axon pathfinding, it is most likely that it functions as a receptor to sense guidance cues rather than as a molecule affecting actin cytoskeletal rearrangement. The data here hint that Dg and Nrk could be two receptors integral to transferring signals important for neuronal layering (Marrone, 2011).

It is concluded that dynamic rearrangement of the actin cytoskeleton is crucial for the central and peripheral nervous system establishment, which depends on proper neuron migration and differentiation. This process requires not only the cell autonomous regulation of neuron motility, but also the interaction between the migrating cell and its underlying substrate. This interaction is often dependent on the signaling transduced via the ECM. The DGC and other factors are believed to be mediators of actin dynamics in growing axons and during neuronal cell morphogenesis, and this study found components that interact with Dys and/or Dg in both of these activities (see The DGC coordinates actin cytoskeleton remodeling). Additionally, disruption in gene expression of these components results in the same phenotypes seen with Dys and Dg mutants in the developing and adult eye. The data lead to the conclusion that the DGC is involved in signaling to cause cytoskeletal rearrangement and actin turnover in growth cones. Since many cases of muscular dystrophies are associated with mental retardation, it is believed that it is important to understand the role of the DGC in axon migration because understanding of this process could aid in finding an adequate therapy for this aspect of the disease's physiology. Since the human brain continues to develop well after gestation, and evidence shows that nerves maintain plasticity throughout an individual's lifespan, therapies could be devised that reverse these defects after birth (Marrone, 2011).

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